A global analysis on the differential regulation of RNA binding proteins (RBPs) by TNF–α as potential modulators of metabolic syndromes
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ABSTRACT:
PROVIDER: PRJNA755604 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15501805_1.fastq.gz | Fastqsanger.gz | |||
| SRR15501805_2.fastq.gz | Fastqsanger.gz | |||
| SRR15501806_1.fastq.gz | Fastqsanger.gz | |||
| SRR15501806_2.fastq.gz | Fastqsanger.gz |
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Project description:Purpose:Metabolic syndrome (MetS) is associated with a group of conditions including diabetes, obesity, insulin resistance etc. The goal of our study is to identify the differentially regulated genes under metabolic syndromes induced by TNF-α. Methods:A Homosapines Reference based Transcriptome sequencing is performed to understand the genes that are diffrerentially regulated under metabolic synromes with TNF-α as upstream. The HEK293 cells were treated with TNF alpha at 10ng/ml for 6hrs to induce the metabolic disturbances mimicking the metabolic syndromes and untreated HEK 293 cells were considered as control. For the Control set (31 million reads) and TNF-α treated (34 million reads) samples was obtained through the Illumina NextSeq 500 platform and comprised of paired end reads of 76 bp (2X76). FastQC software was used to check the quality of the NextSeq 500 paired end reads and were further processed by in-house script for adapter removal and low quality bases trimming. Tophat-2.02 and Cufflinks-2.1.14 tools were used for the analysis. A two fold change and p-value <0.05 cut-off was used to report the differentially expressed genes (DEGs). Owing to the absence of replicates, no FDR correction was used.For each gene a RPKM (Reads per kilo base per million mapped reads) value was calculated for each gene. Apart from the RPKM value, p-value and a fold change value was also calculated for control and treated samples. The genes were validated using qPCR using using Brilliant Ш Ultra-Fast SYBR green qPCR Master Mix in AriaMx PCR System (Agilent Technologies). Results: Around 6056 and 5495 transcripts were shown as up and down regulated respectively using a twofold change cut-off. On excluding the transcripts with no gene names, the numbers of up and down regulated transcripts were found out to be 4444 (3551 genes) and 4365 (3418 genes) respectively.Among which 1218 genes are unique, and highly significant(based on p-value cut-off) and transcriptionally regulated genes by TNF-α, among which about 15% are RBPs; identified by the EuRBPDB data base. Among these transcriptionally regulated genes, we have identified 204 MetS genes by IPA analysis, more than 10% of which are identified as RBPs. Conclusion: Transcriptional regulation of the MetS genes by TNF-α, which also act as RBPs predicts the existence of feed-forward loops and interconnected pathways, which may influence the onset of MetS at the molecular level.
2022-01-21 | GSE182259 | GEO
Project description:Metabolic syndrome (MetS) is associated with a group of conditions, which enhances the risk of diabetes, heart diseases and stroke in the affected individuals. Earlier reports from our lab have shown that Tumor necrosis factor-α (TNF-α) significantly modulates the expression of 56 genes at the alternative splicing level which are involved in various signaling and metabolic pathways (MetS genes) connected to MetS. These MetS genes were predicted to interact with various RNA-binding proteins (RBPs) when exposed to TNF-α, resulting changes in their alternative splicing patterns. Here we are presenting data of an RNA-Seq analysis, which identified 1218 unique, and significantly regulated genes by TNF-α, 15% of which are RBPs . Among the 1218 genes, 204 genes have been identified as MetS genes by the ingenuity pathway analysis, and 10% of the MetS genes are found as RBPs. Our results also show that TNF-α changes the phosphorylation status of certain RBPs such as SR proteins, crucial players in alternative splicing, possibly via changing the activation status of certain upstream signaling molecules which also act as upstream kinases for these proteins. Taken together, these findings suggest that TNF-α influences the regulation of the RBPs at the various levels for their expression, which may lead to the alteration of the splicing pattern of the MetS genes. MetS genes acting as RBPs and are modulated by TNF-α, predict the existence of highly interconnected mechanisms which require further analysis to understand their dual roles on the onset of these diseases.
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Project description:The proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined, central role and mutations promote malignant transformation. More recently, studies have illuminated the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between post-transcriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the post-transcriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a versus RUNX1b/c) that mediate HSC self-renewal versus differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detecting changes in RNA-processing. We utilized this reporter in a CRISPR screen consisting of single guide-RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the post-transcriptional regulation of a key hematopoietic transcription factor and identifies RBPs which may have a widespread, important function in hematopoiesis.
2021-03-16 | GSE145968 | GEO
Project description:Drug serum concentrations will be measured at several time-points for inflammatory disease patients treated with anti-TNF agents. The purpose is to determine which patients that will clinically benefit from either discontinue treatment, adjusting the dose, switch to another anti-TNF agent or a different class of medication.
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Project description:Endothelial-mesenchymal-transition (EndMT) is an important source of cancer-associated fibroblasts (CAFs), which are known to facilitate tumor progression. We have previously shown that EndMT is present in pancreatic tumors and that deficiency of the Tie1 receptor induces EndMT in human endothelial cells. Pancreatic tumors are characterized by the presence of tumor necrosis factor-α (TNF-α). We now show that TNF-α strongly induces human endothelial cells to undergo EndMT. In order to know the secretory feature of cells which undergo EndMT by TNF-α, we conducted a comparative analysis of HMVEC secretome treated or not for 24h and 48h with TNF-α. Secretome study shows that cells treated with TNF-α have an important fibroblast-like secretory capacity, and a proinflamatory signature. Moreover, Ingenuity Pathway Analysis (IPA) shows that pathways implicated in migration, inflammation and fibrosis are predicted to be activated and that necrosis and apoptosis pathways are inhibited. Accordingly cell survival, viability and cycle progression are activated. We show that TNF-α- treated cells secrete proteins related to 16 protumoral pathways, confirming their fibroblastic characteristic. Finally, among the predicted upstream regulators activated, IPA analysis shows that, TNFSF12 and its receptor are present at hight levels in PDAC patients. Altogether these results show the fibroblastic characteristic of treated cells and demonstrate that TNF-α induces CAFs.
2021-09-08 | PXD014488 | Pride
Project description:Deciphering mRNA binding determinants of RBPs
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Project description:G-protein coupled receptors (GPCRs) have diverse roles in physiological processes, including immunity. Gs-coupled GPCRs increase while Gi-coupled ones decrease intracellular cAMP. Previous studies suggest that, in epithelial cells, Gs-coupled GPCRs enhance whereas Gi-coupled GPCRs suppress pro-inflammatory immune responses. In order to examine the issue, we chose beta2 adrenergic receptor and GPR40 as representatives of Gs- and Gi- coupled GPCRs, respectively, and examined their effects on TNF-alpha and IFN-gamma-(TNF-alpha + IFN-gamma) induced gene expression by HaCaT. We used microarrays to detail the global changes of gene expression induced by a beta2 adrenergic receptor agonist terbutaline or GPR40 agonist GW9508 pre-treatment in TNF-alpha + IFN-gamma - stimulated HaCaT cells. HaCaT cells were pre-treated with terbutaline or GW9508, TNF-alpha + IFN-gamma were then added, and cultured for another 24 h. Cells were then used for RNA extraction and hybridization on Affymetrix microarrays. We sought to clarify changes in gene expression after 1) TNF-alpha + IFN-gamma, 2) TNF-alpha + IFN-gamma + terbutaline, and 3) TNF-alpha + IFN-gamma + GW9508 treatment. To this end, we set 4 groups of samples; 1) unstimulated group, 2) TNF-alpha + IFN-gamma-stimulated group, 3) TNF-alpha + IFN-gamma + terbutaline-stimulated group, and 4) TNF-alpha + IFN-gamma + GW9508-stimulated group. In each group, HaCaT cells were stimulated in triplicate wells (n=3).
2010-12-01 | E-GEOD-20297 | biostudies-arrayexpress
Project description:Glycoproteomics, TNF-alpha, membrane proteomics, insulin resistance, adipocyte, SILAC Insulin resistance (IR) is a complex process arising from both environmental and genetic perturbations and leads to a variety of diseases including type-2 diabetes (T2D). The expansion of adipose tissue during obesity results in secretion of proinflammatory cytokines which significantly impairs insulin sensitivity throughout the entire body. Inflammation modulates glycosylation in a variety of cell types however, an investigation of changes in glycosylation following inflammation-induced IR in adipocytes has not been performed. In the present study, we performed a quantitative N-glycomic analysis of TNF-alpha induced IR in adipocytes and identified the regulation of specific N-glycans including an increase in terminal di-galactose- and decrease in di-sialic acid-containing glycans with alpha-2,3 linkages. Quantitative analysis of the membrane-associated proteome in TNF-alpha treated adipocytes identified the regulation of specific glycosyltransferases and glycosidases which correlated with the regulated N-glycans. This included the up- and down-regulation of B4GalT5 and ST3Gal6, respectively at both the protein and mRNA level. To identify the protein and glycosylation sites modified with these regulated N-glycans, we performed a site-specific quantitative glycoproteomic analysis of enriched N-glycopeptides from TNF-alpha treated adipocytes with and without deglycosylation. The combined workflow provided a relative quantification of changes in protein abundance verses N-glycosylation occupancy verses site-specific N-glycans on a proteome-wide level. This revealed the modulation of a subset of N-glycan compositions on specific proteins including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane. Finally, knockdown of B4GalT5 with siRNA and analysis of released N-glycans resulted in the down-regulation of di-galactose containing glycans, confirming the involvement of this enzyme in the TNF-alpha regulated N-glycome.
2015-11-09 | PXD002435 | Pride
Project description:We completed genome-wide microarray analysis in Sirt6 knockout (ko) mouse embryonic fibroblasts (MEFs) and control wildtype (wt) MEFs. Prior to RNA extraction, MEFs were treated with TNF-alpha (10 ng/ml) for the indicated times. Total RNA was extracted with TRIzol (Invitrogen) and amplified with Ambion Amino Allyl MessageAmp II Amplification kit. Amplified mouse Universal Reference RNA (Stratagene) was used as reference RNA for analysis. Array hybridization of mouse MEEBO arrays is as described (Hendrickson et al., PLoS ONE, 2008). Groups of assays that are related as part of a time series. Compound Based Treatment: 10 ng/ml TNF-alpha Keywords: time_series_design Computed
2010-06-24 | E-GEOD-13207 | biostudies-arrayexpress
Project description:We established HeLa cell lines stably expressing a short hairpin RNA against SIRT6 (S6 sh2), and then completed genome-wide microarray analysis in shSIRT6 knock-down and control pSR HeLa cells. Prior to RNA extraction, HeLa cells were treated with TNF-alpha (5 ng/ml) for the indicated times. Total RNA was extracted with TRIzol (Invitrogen) and amplified with Ambion MessageAmp RNA Amplification kit. Amplified human Universal Reference RNA (Stratagene) was used as reference RNA for analysis. Construction and array hybridization of human cDNA microarrays were performed as previously described (Perou et al., 2000). Groups of assays that are related as part of a time series. Compound Based Treatment: 5 ng/ml TNF-alpha Keywords: time_series_design Computed
2010-06-24 | E-GEOD-13206 | biostudies-arrayexpress